Gas-Chromatography/Mass Selective Detector를 사용하여 쥐의 뇨시료 중 benzidine 대사체의 확인 및 in vitro 독성

Identification of Benzidine Metabolites in Rats by Gas Chromatography/Mass Selective Detector and its Toxicity in vitro

Metabolism study of the dye, benzidine, was performed by gas chromatography-mass selective detector (GC/MSD) in the urine of rats orally administered 100mg/kg benzidine. Urine samples were collected in metabolic cages for 0-24, 24-48, and 48-72 hrs. Ten ml of the urine was extracted with XAD-2 resin and the XAD-2 column was eluted with methanol. After evaporation, benzidine and its metabolites were extracted with diethyl ether (for non-conjugated fraction). For conjugated metabolites, beta-glucuronidase was added to the aqueous layer that was incubated for 1hr at 50oC and the aqueous layer was extracted as in non-conjugated fraction. Aliquot of trimethylsilylated derivatives was applied to the GC/MSD. The mutagenicity of benzidine and its acetylated metabolites was tested by histidine/reversion assay. Five metabolites observed and confirmed either by electron impact and chemical ionization modes of the GC/MSD, or authentic compounds were monoacetyl-, diacetyl-, hydroxyacetyl-, hydroxydiacetyl-, and hydroxy-benzidine. Monoacetyl-benzidine was more potent than benzidine in histidine/reversion assay. This data indicates that monoacetylation of benzidine may be one of the metabolites produced in metabolic activation process.